The present invention relates to genetically engineered L-sorbose reductase-deficient mutants of a microorganism belonging to the genus Gluconobacter or Acetobacter. The present invention also relates to the production of L-sorbose by fermentation as well as to the production of vitamin C.
The production of vitamin C has been conducted by Reichstein method which involves a fermentation process for the conversion from D-sorbitol to L-sorbose by a microorganism belonging to the genus Gluconobacter or Acetobacter, as a sole biological step. The said conversion to L-sorbose is one of the key steps for the efficiency of vitamin C production. It was, however, observed that the product, L-sorbose, was consumed after the consumption of the substrate, D-sorbitol, during the oxidative fermentation with the said microorganism. This phenomenon was understood that L-sorbose was reduced by NADPH-linked L-sorbose reductase (hereinafter occasionally referred to as SR) present in the cytosol (See: Sugisawa et al., Agric. Biol. Chem. 55: 2043-2049, 1991). It was reported that, in Gluconobacter, D-sorbitol was converted to D-fructose which could be incorporated into the pentose pathway and further metabolized to C02 (Shinjoh et al., Agric. Biol. Chem. 54: 2257-2263, 1990). Such pathways consuming the product as well as the substrate might have caused less productivity of vitamin C ultimately.
An improvement of L-sorbose production was reported by Nogami et al. in Japanese Patent Application Kokai No. 51054/1995. They subjected the microorganisms of Gluconobacter oxydans and Gluconobacter suboxydans to conventional chemical mutagenesis and isolated the mutant strains whose ability of utilizing D-sorbitol as a sole assimilable carbon source was reduced. By applying such mutant to the fermentation for L-sorbose production, they observed more than 2xcx9c3% improvement of the productivity in comparison with the productivity of the parent strain.
One of the disadvantages which is often observed in mutant strains produced by the conventional mutagenesis is back mutation which nullifies the improved characteristics of the mutant strains during the course of fermentation or subculture of the mutant, which would result in decreased productivity of vitamin C ultimately. Therefore a stably mutated strain with respect to L-sorbose reductase is desired.
The present invention provides a genetically engineered microorganism belonging to the genus Gluconobacter or Acetobacter, which has an engineered gene for the biological activity of reducing L-sorbose which is more than 90% non-functional in developing the said biological acitivity. The engineered microorganism is derived from a microorganism belonging to the genus Gluconobacter or Acetobacter, and has the biological activity for reducing L-sorbose that is less than 10% of the amount of the activity of the wild type organism.
The present invention also provides a method for producing L-sorbose by incubating the genetically engineered microorganism in a medium, and obtaining L-sorbose from the medium. L-Sorbose produced in this manner can be used in the manufacture of vitamin C.
The present invention further provides a method for producing the genetically engineered microorganism by mutating the gene of a microorganism of the genus Gluconobacter or Acetobacter which encodes a protein having the L-sorbose reductase activity, determining the L-sorbose reductase activity of the resulting microorganism which has the mutated gene and selecting the genetically engineered microorganism having L-sorbose reductase activity that is less than 10% of the amount of the activity of the wild type microorganism.